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Biomedical subjects

Michael S Pepper

Publications and source records attributed to Michael S Pepper.

31 records · Page 2Linked to original sources
PubMed2003

Vascular endothelial growth factor (VEGF) receptor-2 signaling mediates VEGF-C(deltaNdeltaC)- and VEGF-A-induced angiogenesis in vitro.

Angiogenesis and lymphangiogenesis are regulated by members of the vascular endothelial growth factor (VEGF) family of cytokines, which mediate their effects via tyrosine kinase VEGF receptors -1, -2, and -3. We have used wild-type and mutant forms of VEGFs -A, -B, and -C, a pan-VEGFR tyrosine kinase inhibitor (SU5416) as well as neutralizing anti-VEGFR-2 antibodies, to determine which VEGF receptor(s) are required for bovine endothelial cell invasion and tube formation in vitro. This was compared to the ability of these cytokines to induce expression of members of the plasminogen activator (PA)-plasmin system. We found that cytokines which bind VEGFR-2 (human VEGF-A, human VFM23A, human VEGF-C(deltaNdeltaC), and rat VEGF-C(152)) induced invasion, tube formation, urokinase-type-PA, tissue-type-PA, and PA inhibitor-1, invasion and tube formation as well as signaling via the MAP kinase pathway were efficiently blocked by SU5416 and anti-VEGFR-2 antibodies. In contrast, cytokines and mutants which exclusively bind VEGFR-1 (human VFM17 and human VEGF-B) had no effect on invasion and tube formation or on the regulation of gene expression. We were unable to identify cytokines which selectively stimulate bovine VEGFR-3 in our system. Taken together, these findings point to the central role of VEGFR-2 in the angiogenic signaling pathways induced by VEGF-C(deltaNdeltaC) and VEGF-A.

Animals↗
PubMed2003

Ets 1 is expressed in capillary blood vessels but not in lymphatics.

Little is known about the mechanisms that regulate lymphangiogenesis and, in particular, about regulation at the transcriptional level. To determine whether parallels exist in the mechanisms of angiogenesis and lymphangiogenesis, the expression of the Ets 1 transcription factor (which has previously been shown to be involved in angiogenesis) was examined in two transgenic mouse lines, namely RipVEGF-C mice (which develop lymphatic capillaries ectopically around islets of Langerhans) and Rip1Tag2 mice (which develop insulinomas that are not lymphangiogenic). Crossing the two lines results in double transgenic mice that develop lymphatics around insulinomas, which in turn promotes metastasis to regional lymph nodes. By immunohistochemistry, it was found that, in contrast to blood vessels, lymphatic vessels in wild-type and transgenic mice did not express Ets 1. Immunohistochemical staining for matrix metalloproteinase (MMP)-2 and MMP-9 as well as membrane type 1-MMP (MT1-MMP/MMP-14), all of which are encoded by known or potential Ets 1 target genes, showed the same cellular distribution as Ets 1. These findings suggest that the transcriptional regulatory mechanisms of lymphangiogenesis differ from those involved in angiogenesis. Furthermore, if proteases are involved in lymphangiogenesis, these observations suggest that they are different from those considered to be important for blood vessel formation.

Animals↗
PubMed2003

Expression of vascular endothelial growth factor (VEGF)-C and VEGF-D, and their receptor VEGFR-3, during different stages of cervical carcinogenesis.

Cervical carcinogenesis has well-defined stages of disease progression including three grades of pre-invasive lesions--cervical intraepithelial neoplasia grades 1-3 (CIN 1-3)--and invasive cervical cancer. However, the biological properties of CIN lesions prone to develop invasive disease are not well defined. Recent observations suggest that early invasive disease spreads to regional lymph nodes in several tumour types and that growth factors (VEGF-C and VEGF-D) involved in new lymphatic vessel formation may play a crucial role in this process. The present study has assessed the expression of VEGF-C and VEGF-D, and their receptor VEGFR-3, in 152 cervical lesions (33 CIN 1, 33 CIN 2, 37 CIN 3, and 49 squamous cell carcinomas) to determine whether expression of lymphangiogenic factors occurs prior to invasion. The presence of lymphatic vessels was determined using LYVE-1 and podoplanin staining, as well as double immunostaining for LYVE-1/CD34 and podoplanin/CD34. In situ hybridization was performed to determine VEGFR-3 mRNA expression. A significant positive correlation was found between VEGF-C, VEGF-D, and VEGFR-3 expression through the different stages of cervical carcinogenesis. Significant differences in protein expression for VEGF-C, VEGF-D, and VEGFR-3 were found between CIN 1-2 and CIN 3 (p<0.001 for all), but not between CIN 3 and cervical cancer. More than 50% of the CIN 3 lesions showed moderate to strong staining for VEGF-C and VEGF-D, whereas most of the early pre-cancerous lesions (CIN 1 and 2) were negative. In cervical cancer, similar observations to those in CIN 3 were found. VEGFR-3 mRNA expression was found in the cytoplasm of epithelial neoplastic cells and VEGFR3 protein expression was found in more than 50% of CIN 3 lesions and cervical cancers, compared with 15% in CIN 1 and 2. These findings suggest an autocrine growth stimulation pattern via VEGFR-3. Adjacent CIN 3 was present in nine cervical cancers and displayed strong expression for VEGF-C, VEGF-D, and VEGFR-3. These results suggest that in cervical carcinogenesis a switch to the lymphangiogenic phenotype may occur at the stage of CIN 3.

Adolescent↗
PubMed2003

alphav beta 3 and alphav beta 5 integrin antagonists inhibit angiogenesis in vitro.

Although angiogenesis is believed to require cell-extracellular matrix interactions which are mediated in part via integrins alphav beta 3 and alphav beta 5, a formal demonstration that alphav beta 3 and alphav beta 5 are involved in endothelial-cell invasion and capillary-like tube formation is still required. This has arisen from the cellular complexities which occur in vivo and the difficulty in finding appropriate in vitro model systems. Here we have used a three-dimensional assay which employs bovine aortic and microvascular endothelial cells, to show that alphav beta 3 and alphav beta 5 regulate angiogenesis in vitro. We cloned and characterized 350-450 bp regions of the bovine homologues of alphav, beta 3 and beta 5, covering much of the beta -propeller and A-domain regions, and show that they are >95% identical to their human orthologues. We used cyclic peptides EMD 121974, 85189 and 66203, which selectively inhibit alphav beta 3 and alphav beta 5, but not gpIIbIIIa or alpha5 beta 1, to probe in vitro angiogenesis induced by angiogenic cytokines in three-dimensional fibrin or collagen gels. We found that these peptides are potent inhibitors of endothelial cell invasion and differentiation induced by vascular endothelial growth factor-A or fibroblast growth factor-2 but do not affect the unstimulated cells in 3D culture. Inhibition was greatest when cells were grown on fibrin, but also occurred on collagen I which is not a recognized ligand for alphav beta 3. These findings demonstrate the requirement for endothelial cell alphav beta 3 and alphav beta 5 integrins during angiogenesis in vitro, and are in accord with the proposed therapeutic application of alphav beta 3 and alphav beta 5 antagonists.

Angiogenesis Inhibitors↗
PubMed2003

Lymphangiogenesis and tumor metastasis.

In several human cancers, increased expression in primary tumors of vascular endothelial growth factor-C (VEGF-C) is correlated with regional lymph node metastasis. Studies using transgenic mice overexpressing VEGF-C, or xenotransplantation of VEGF-C-expressing tumor cells into immunodeficient mice, have demonstrated a role for VEGF-C in tumor lymphangiogenesis and the subsequent formation of lymph node metastasis. However, at variance with data obtained in animal models, there is at present very little evidence for lymphangiogenesis in human tumors. Nonetheless, the striking correlation between levels of VEGF-C in primary human tumors and lymph node metastases exists, which suggests that VEGF-C may serve functions other than lymphangiogenesis. Thus, VEGF-C may activate pre-existing lymphatics which in turn become directly involved in tumor cell chemotaxis, intralymphatic intravasation and distal dissemination. A reciprocal dialogue is therefore likely to exist between tumor and lymphatic endothelial cells which results in the formation of lymph node metastases.

Angiogenic Proteins↗
PubMed2002

Molecular characterization of lymphatic endothelial cells.

The lymphatic microvasculature is uniquely adapted for the continuous removal of interstitial fluid and proteins and is an important entry point for leukocytes and tumor cells. Specialized functions of lymphatics suggest differences in the molecular composition of the lymphatic and blood vascular endothelium. However, the extent to which the two cell types differ is still unclear, and few molecules that are truly specific to lymphatic endothelial cells have been identified to date. We have isolated primary lymphatic and blood microvascular endothelial cells from human skin by immunoselection with the lymphatic marker LYVE-1 and demonstrate that the two cell lineages express distinct sets of vascular markers and respond differently to growth factors and extracellular matrix. Comparative microarray analysis of gene-expression profiles revealed a number of unique molecular properties that distinguish lymphatic and blood vascular endothelium. The molecular profile of lymphatic endothelium seems to reflect characteristic functional and structural features of the lymphatic capillaries. Classification of the differentially expressed genes into functional groups revealed particularly high levels of genes implicated in protein sorting and trafficking, indicating a more active role of lymphatic endothelium in uptake and transport of molecules than previously anticipated. The identification of a large number of genes selectively expressed by lymphatic endothelium should facilitate the discovery of hitherto unknown lymphatic vessel markers and provide a basis for the analysis of the molecular mechanisms accounting for the characteristic functions of lymphatic capillaries.

Biomarkers↗
PubMed2002

Mesenchymal cells potentiate vascular endothelial growth factor-induced angiogenesis in vitro.

The role of soluble factors (including angiogenic cytokines) and extracellular matrix components in the regulation of angiogenesis is clearly established. However, the interrelationship between these factors and perivascular mesenchymal cells is not well understood. Here we have used a three-dimensional collagen gel coculture system to assess the effect of mesenchymal C3H10T1/2 cells on vascular endothelial growth factor-A (VEGF-A)- and fibroblast growth factor-2 (FGF-2)-induced angiogenesis in vitro. We found that coculture markedly potentiated the angiogenic activity of VEGF-A, irrespective of whether or not direct cell-to-cell contact occurred. In contrast, under conditions in which cell-to-cell contact was possible, FGF-2-induced angiogenesis was inhibited by cocultured 10T1/2 cells; this effect was not seen when cell-to-cell contact was prevented. Attempts to identify the molecules responsible for this effect allowed us to exclude FGF-2, transforming growth factorbeta1, platelet derived growth factor-BB, angiopoietin-1, and NO as possible mediators of the potentiating effect of coculture on VEGF-A-induced invasion. In the living organism, angiogenesis occurs in a three-dimensional microenvironment. Contrary to the inhibitory effect of 10T1/2 cells previously reported by others in two-dimensional cultures, our data demonstrate that the paracrine interaction between endothelial and mesenchymal cells potentiates angiogenesis in vitro and that this is cytokine-specific, i.e., it occurs with VEGF-A but not with FGF-2.

Angiogenesis Inducing Agents↗
PubMed2002

Heterogeneity of smooth muscle cell populations cultured from pig coronary artery.

OBJECTIVE: Heterogeneous smooth muscle cell (SMC) populations have been described in the arteries of several species. We have investigated whether SMC heterogeneity is present in the porcine coronary artery, which is widely used as a model of restenosis. METHODS AND RESULTS: By using 2 isolation methods, distinct medial populations were identified: spindle-shaped SMCs (S-SMCs) after enzymatic digestion, with a "hill-and-valley" growth pattern, and rhomboid SMCs (R-SMCs) after explantation, which grow as a monolayer. Moreover, the intimal thickening that was induced after stent implantation yielded a large proportion of R-SMCs. R-SMCs exhibited high proliferative and migratory activities and high urokinase activity and were poorly differentiated compared with S-SMCs. Heparin and transforming growth factor-beta2 inhibited proliferation and increased differentiation in both populations, whereas fibroblast growth factor-2 and platelet-derived growth factor-BB had the opposite effect. In addition, S-SMCs treated with fibroblast growth factor-2 or platelet-derived growth factor-BB or placed in coculture with coronary artery endothelial cells acquired a rhomboid phenotype. This change was reversible and was also observed with S-SMC clones, suggesting that it depends on phenotypic modulation rather than on selection. CONCLUSIONS: Our results show that 2 distinct SMC subpopulations can be recovered from the pig coronary artery media. The study of these subpopulations will be useful for understanding the mechanisms of restenosis.

Animals↗
PubMed2002

Overexpression of vascular endothelial growth factor-A165 enhances tumor angiogenesis but not metastasis during beta-cell carcinogenesis.

The pivotal role of vascular endothelial growth factor (VEGF-A) in the regulation of angiogenesis, in particular in the onset and maintenance of tumor angiogenesis, has been demonstrated repeatedly in experimental model systems and, more recently, in clinical trials. Experimental evidence has also suggested that up-regulated expression of VEGF-A may cooperate with other genetic or epigenetic changes to induce or accelerate tumor progression to invasive and metastatic cancers. Here we report the generation of transgenic mouse lines that express human VEGF-A165 under the control of the rat insulin promoter in the beta cells of pancreatic islets of Langerhans (Rip1VEGF-A). These mice do not exhibit detectable changes in islet development, vascularization, or physiology. Intercrosses of these mice with a transgenic mouse model of pancreatic beta cell carcinogenesis (Rip1Tag2) result in an earlier onset of tumor angiogenesis and with it accelerated tumor growth and mortality. The transition from benign tumors (adenoma) to malignant tumors (carcinoma) is modestly accelerated; however, tumor metastases are not observed. Our findings indicate that in beta-cell tumorigenesis, overexpression of VEGF-A165 accelerates the onset of tumor angiogenesis and with it tumor progression but is not sufficient to induce tumor metastasis.

Animals↗
PubMed2002

Lymphatic dissemination of tumour cells and the formation of micrometastases.

Most human cancers show evidence of metastatic spread to regional lymph nodes, and the extent of lymph-node involvement is directly related to dinical outcome. Increased expression of vascular endothelial growth factor C in primary tumours is associated with increased dissemination of tumour cells to regional lymph nodes in various human carcinomas. Arguments favouring the activation of pre-existing lymphatic endothelium and the de novo formation of lymphatic capillaries (lymphangiogenesis) are discussed. We highlight recent advances in the molecular detection and characterisation of lymph-node micrometastases, as well as potential microenvironmental factors, such as chemokines, which may influence the migration and growth of metastatic tumour cells. Finally, we examine the clinical significance of lymphatic-mediated tumour-cell dissemination and the formation of lymph-node micrometastases.

Cell Movement↗
PubMed2002

Microscopy: an art?

There can be no doubt that the finest creator of beauty is Mother Nature. And in many ways, science is the exploration of this beauty and of the mechanisms that have created it. Microscopy, as a technique in scientific investigation, has had a key role in uncovering nature's beauty, which has led some to propose that microscopy could be described as an art or even an art form. But is this claim justified?

Anatomy, Artistic↗
PubMed2002

Increased angiostatin levels in bronchoalveolar lavage fluids from ARDS patients and from human volunteers after lung instillation of endotoxin.

Acute respiratory distress syndrome (ARDS) is characterized by a disruption of the alveolar-capillary barrier, due to both an epithelial and an endothelial dysfunction. Whereas epithelial apoptosis seems to be mainly mediated by Fas ligand, the mediators of endothelial damage remain to be identified. Angiostatin, a powerful inhibitor of angiogenesis in vivo, also specifically induces apoptosis in endothelial cells. The concentration of various enzymes that cleave angiostatin from plasminogen was reported to be significantly increased in bronchalveolar lavage (BAL) fluids from patients with ARDS. Therefore, in this study, we investigated whether angiostatin was generated during the pulmonary inflammatory response of both healthy subjects challenged with endobronchial endotoxin and in patients with ARDS. We found significantly elevated angiostatin levels in BAL fluids from patients at risk for and with early ARDS (up to 0.022% and 0.018% of total protein, respectively), as well as in BAL fluids from volunteers treated with endotoxin (up to 1.17% of total protein), as compared to BAL fluids from control patients (< 0.005% of total protein). These data suggest that angiostatin may contribute to the endothelial damage observed in ARDS, probably via an increased permeability of the alveolar capillary barrier, allowing for an intra-alveolar processing of its precursor plasminogen.

Adult↗